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The Journal of Immunology, 2008, 180, 2419-2428
Copyright © 2008 by The American Association of Immunologists, Inc.
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Integrin β1 Regulates Phagosome Maturation in Macrophages through Rac Expression1

Qing-Qing Wang*, Hong Li*, Tim Oliver{dagger}, Michael Glogauer{ddagger}, Jian Guo* and You-Wen He2,*

Departments of * Immunology and {dagger} Cell Biology, Duke University Medical Center, Durham, NC 27710; and {ddagger} Canadian Institute of Health Research, Group in Matrix Dynamics, Faculty of Dentistry, University of Toronto, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Phagocytosis and subsequent phagosome maturation by professional phagocytes are essential in the clearance of infectious microbial pathogens. The molecular regulation of phagosome maturation is largely unknown. We show that integrin β1 plays critical roles in the phagocytosis of microbial pathogens and phagosome maturation. Macrophages lacking integrin β1 expression exhibit reduced phagocytosis of bacteria, including group B streptococcus and Staphylococcus aureus. Furthermore, phagosomes from macrophages lacking integrin β1 show lowered maturation rate, defective acquisition of lysosome membrane markers, and reduced F-actin accumulation in the periphagosomal region. Integrin β1-deficient macrophages exhibit impaired bactericidal activity. We found that the expression of the Rho family GTPases Rac1, Rac2, and Cdc42 was reduced in integrin β1-deficient macrophages. Ectopic expression of Rac1, but not Cdc42, in integrin β1-deficient macrophages restored defective phagosome maturation and F-actin accumulation in the periphagosomal region. Importantly, macrophages lacking Rac1/2 also exhibit defective maturation of phagosomes derived from opsonized Escherichia coli or IgG beads. Taken together, these results suggest that integrin β1 regulates phagosome maturation in macrophages through Rac expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Phagocytes such as macrophages use a variety of phagocytic receptors, including IgG FcRs (Fc{gamma}R), complement receptors, scavenger receptors, integrins, and TLRs to internalize microbes (1). Upon binding of opsonized particles to phagocytic receptors, a membrane structure termed phagocytic cup is formed for particle internalization. Subsequently, phagocytic cups sealed by the cytoplasmic membrane form intracellular vacuoles called phagosomes (2, 3). With time, phagosomes undergo dramatic changes in composition by fusing with early endosomes, late endosomes, and lysosomes, a process described as phagosome maturation. Phagosome maturation in macrophages results in the killing of internalized microorganisms with complete degradation and clearance of phagocytic cargo (4, 5, 6).

The molecular mechanism underlying phagocytosis has been studied extensively. It is well established that members of the Rho GTPase family provide critical signals for phagocytic cup formation during FcR-mediated phagocytosis (7, 8, 9, 10, 11). However, the signals that control phagosome maturation remain to be fully elucidated. Underhill et al. (12) demonstrated TLR2 recruitment to phagosomes, suggesting that TLRs may play a role in phagosome maturation. However, subsequent studies examining the function of TLRs in phagosome maturation generated conflicting results. In one study, macrophages lacking TLR4 expression exhibited accelerated fusion between endosomes/lysosomes and phagosomes containing opsonized cells or particles (13). In another study, TLRs and MyD88-deficient macrophages exhibited delayed phagosome maturation when Escherichia coli or Staphylococcus aureus was phagocytosed (14). A recent report demonstrated that phagosome maturation proceeds normally in the absence of TLR2 and TLR4 stimulation (15).

Integrins are transmembrane receptors composed of {alpha} and β heterodimers. The integrin family is involved in cell-cell and cell-extracellular matrix interactions in many cell types (16, 17). Macrophages express various integrins, including {alpha}Mβ2 (CD18/CD11b), {alpha}4β1 (CD49d/CD29), and {alpha}5β1 (CD49e/CD29). The role of integrin {alpha}Mβ2 (CD18/CD11b) as a phagocytic receptor through the binding of iC3b-opsonized particles has been well established (3). However, the function of other two integrins {alpha}4β1 and {alpha}5β1 in phagocytosis remains to be directly examined in cells lacking the expression of these integrins. Our recent studies have identified that integrin {alpha}4β1 and {alpha}5β1 serve as receptors for the pattern recognition molecule mindin (18, 19), suggesting that these integrins may play critical roles in macrophage clearance of microbial pathogens.

To determine the role of integrin {alpha}4β1 and {alpha}5β1 in macrophage phagocytosis, we generated mice lacking integrin β1 expression specifically in macrophages by crossing mice with floxed integrin β1 alleles (20) to transgenic mice expressing a Cre recombinase under the promoter of lysozyme M (21). Macrophages lacking integrin β1 expression exhibit defective phagocytosis of group B streptococcus (GBS)3 and S. aureus, but not E. coli. Furthermore, integrin β1-deficient macrophages also show defective phagosome maturation after internalization of opsonized E. coli and impaired bactericidal activity. This defective phagosome maturation was most likely due to reduced expression of the Rho GTPase Rac1 and Rac2 in integrin β1-deficient macrophages. Although the expression of Rac1, Rac2, and Cdc42 was reduced in integrin β1-deficient macrophages, ectopic expression of Rac1, but not Cdc42, restored the defective phagosome maturation and impaired periphagosomal F-actin accumulation in integrin β1-deficient macrophages. In addition, macrophages lacking both Rac1 and Rac2 expression also exhibited defective phagosome maturation. Collectively, our results indicate that integrin β1 is involved in the phagocytosis of certain bacteria and critically regulates phagosome maturation through Rac expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

CD29 (β1 integrin) floxed (CD29f/f) mice (20) were obtained from The Jackson Laboratory. Mice lacking both Rac1 and Rac2 expression in macrophages were generated by crossing Rac1f/-LysM-Cre mice to Rac2–/– mice, as previously described (22). To generate β1 integrin-deficient macrophages, CD29f/f mice were crossed with LysM-Cre transgenic mice (21). All mice were maintained in a specific pathogen-free facility at the Duke University Vivarium. Animal usage was conducted according to protocols approved by the Duke University Institutional Animal Care and Use Committee. All mice were used at 6–12 wk of age.

Reagents

Fluorescence-labeled mAbs, including anti-CD11b cyanin 5/PE or FITC, CD29 FITC, CD18 FITC, CD11b FITC, human CD2 (hCD2) FITC, CD49d PE, CD49e PE, and unlabeled mAbs against Rac 1, Rac2, Cdc42, lysosomal-associated membrane protein (LAMP)-1, LAMP-2, and Rab5, were obtained from eBioscience, Biolegend, or Santa Cruz Biotechnology. GBS was a clinical isolate provided by J. Wright (Duke University Medical Center, Durham, NC). E. coli, S. aureus, and Salmonella were from American Type Culture Collection (ATCC 11105, 12598, 35987).

Macrophage phagocytosis

Bone marrow-derived macrophages (BMM{phi}) were generated from 7-day cultures of bone marrow cells of CD29f/f, CD29f/fLysM-Cre, or Rac1f/-LysM-Cre.Rac2–/– mice in DMEM containing 20% FBS and 30% L929 cell-conditioned medium. CD29-deficient macrophages were purified by FACS sorting based on CD29 surface expression. For confocal microscopy and fluorescence resonance energy transfer (FRET) analysis, fully differentiated macrophages were transferred to petri dishes containing sterile glass coverslips and left for 18 h to allow a confluent monolayer to establish.

Macrophage phagocytosis assay was performed, as described (11). For analysis of Fc{gamma}R-mediated phagocytosis, BMM{phi} were cultured overnight on glass coverslips and incubated with human IgG-coupled latex beads (diameter 3 µM; Sigma-Aldrich) or SRBCs opsonized with rabbit anti-SRBC IgG (1 mg/ml; Nordic Immunology) at a ratio of 1:10. Samples were incubated for 15–30 min at 4°C for binding, followed by 15–30 min at 37°C for phagocytosis. Cell surface-bound SRBCs were lysed with hypotonic buffer, and the macrophages were fixed in 2.5% glutaraldehyde solution before being counted.

Complement-mediated phagocytosis was performed using FITC-labeled zymosan particles (Molecular Probes). Zymosan particles (5.4 x 107) were opsonized with 150 µl of normal mouse serum at 37°C for 30 min and mixed with macrophages at different ratios. The mixtures were then centrifuged and incubated at 4°C for 30 min to allow zymosan binding. Unbound particles were washed with cold PBS. Prewarmed medium was added to initiate phagocytosis. Macrophages were fixed with 3.7% paraformaldehyde after cell surface-bound zymosan particles were quenched by trypan blue (2 mg/ml). Phagocytosed FITC-zymosan particles were counted under a fluorescence microscope.

To analyze phagocytosis of bacteria, FITC-labeled bacteria were opsonized with normal mouse serum, mixed with BMM{phi} in suspension at a ratio of 1:100 (cell/bacteria), and rotated for 30 min at 37°C. Unbound bacteria were washed with cold PBS. Macrophages were fixed with 3.7% paraformaldehyde after cell surface-bound bacteria were quenched with trypan blue (2 mg/ml). The fluorescence of the cells was determined by flow cytometry using a BD Biosciences FACScan.

Quantitative assessment of colocalization of internalized bacteria and lysosomes

BMM{phi} were transferred to petri dishes containing sterile glass coverslips and left for 18 h to allow a confluent monolayer to establish. BMM{phi} were incubated in Lysotracker Red (Molecular Probes) for 2.5 h at 37°C, followed by 2 h in fluor-free medium. The cells were mixed with opsonized E. coli labeled with the Alexa Fluor (AF) 488 (Molecular Probes) at a ratio of 1:50 (cell/bacteria), incubated for 20 min at 37°C. Unbound bacteria were washed with cold PBS. After incubation periods of 30, 60, 90, 120, and 150 min at 37°C, cell surface-bound bacteria were quenched with trypan blue (2 mg/ml). Macrophages were fixed with 3.7% paraformaldehyde. To determine the kinetics of phagosome maturation, the colocalization of AF488-labeled E. coli with Lysotracker Red (a marker of acidic compartments) was evaluated by laser confocal microscopy (Leica 510; Leica Microsystems). The percentage of E. coli-containing phagosomes that colocalized with Lysotracker (fluorescence overlay) was quantitatively analyzed using the colocalization dialogue of Metamorph offline software version 7.0 (Universal Imaging, a subsidiary of Molecular Devices) by randomly scanning >10 cells in each test group in two or more independent experiments.

FRET assay

BMM{phi} in petri dishes were incubated in 100 µg/ml AF594 hydrazide (Molecular Probes) for 3.5 h at 37°C, followed by 18 h in fluor-free medium. AF488-labeled E. coli, AF488-human IgG beads, or opsonized FITC-zymosan particles were mixed with macrophages at a ratio of 50:1. Following uptake for 20 min at 37°C, unbound particles were washed with cold PBS. After incubation periods of 30, 60, 90, 120, and 150 min at 37°C, the surface-bound fluorescence was quenched with trypan blue (2 mg/ml). Before microscopy, cells were fixed with 3.7% paraformaldehyde, and samples were preserved in 80% glycerol. The presence of FRET between AF488 (donor) or FITC and AF594 (acceptor) molecules was established using laser confocal microscopy (Leica SP2) and the Leica FRET Acceptor Bleaching wizard, by noting the loss of intensity of donor that accompanies the close molecule proximity of acceptor. Donor fluorescence intensity was recorded in the same sample before and after destroying the acceptor by selectively photobleaching in a carefully defined region of interest. The energy transfer efficiency was quantified as follows: FRETeff = (Dpost – Dpre)/Dpost, where Dpost is the fluorescence intensity of the donor after acceptor photobleaching, and Dpre the fluorescence intensity of the donor before acceptor photobleaching. The FRETeff is considered positive when Dpost > Dpre. The percentage of FRET-positive regions and FRET efficiency values was quantified by randomly counting over 10 areas within one cell and counting >10 cells in each test group in two or more independent experiments.

Staining of F-actin polymerization

Polymerization of cellular actin was measured by a phalloidin-binding assay. After internalization of AF488-labeled E. coli for 30 min, macrophages were fixed in 10% formalin for 20 min at 4°C. The cells were permeabilized for 5 min with 0.1% Triton X-100 in PBS and then stained with 0.2 µM rhodamine-phalloidine (Molecular Probes) for 30 min on ice, and cell images were analyzed with laser confocal microscopy (Leica 510). The percentage of colocalization of pixels occupied by both phagosomes and F-actin was quantitatively analyzed by Metamorph offline software, as described above.

Bacteria-killing assay

BMM{phi} were mixed with opsonized bacteria at a ratio of 1:50, rotated for 15 min at 37°C. Unbound bacteria were removed by washing, and the cells were further incubated for 90 min at 37°C. To release the intracellular bacteria, macrophages were resuspended in 1 ml of PBS with 0.05% (w/v) saponin and the debris was broken up using a ground glass homogenizer. The suspensions were serially diluted in sterile PBS, and 20-µl samples were spread on agar plates. The plates were incubated at 37°C overnight, and the resulting colonies were counted.

Bead-containing phagosome purification and Western blot

Isolation of latex bead-containing phagosomes was performed using a modification of a method described by Desjardins et al. (23). After the pulse and chase of latex beads (60, 120 min) at 37°C, the BMM{phi} were washed in cold PBS and scraped in PBS at 4°C. The cells were washed for 5 min in homogenization buffer (250 mM sucrose and 3 mM imidazole (pH 7.4)) at 4°C, resuspended in 1 ml of homogenization buffer, and homogenized on ice with a Dounce homogenizer. The homogenization was conducted until ~90% of cells were broken without major breakage of the nucleus, as monitored by light microscopy. After centrifugation at 1200 rpm for 5 min at 4°C, the supernatant containing the phagosomes was recovered. The phagosomes were then isolated by flotation on a sucrose gradient, as described (23). Equivalent amounts of phagosome proteins were loaded onto 10% SDS-polyacrylamide gels. LAMP-1, LAMP-2, and Rab5 from bead-containing phagosomes were identified by Western blot.

BMM{phi} stimulated with E. coli at indicated time points were harvested and lysed in cell lysis buffer. The lysates were fractionated by 12% SDS-PAGE gel and transferred onto nitrocellulose membrane. Rac1, Rac2, and Cdc42 proteins were detected by Western blot. To ectopically express Rac1 or Cdc42 in macrophages, the pMI retrovirus vector expressing hCD2 and mouse wild-type (WT) Rac1 and Cdc42 cDNA bicistronically were used to generate retroviruses, as described (24). BMM{phi} were infected with retroviruses at days 2 and 3 of the culture. Infected hCD2+ and CD29+ or CD29– BMM{phi} were sorted by FACS for Western blot, phagocytosis, FRET assay, or F-actin staining.

Peritoneal macrophage assay

Peritoneal macrophages were collected from CD29f/f or CD29f/fLysM-Cre mice 3–4 days following injection of 1 ml of 3% thioglycolate broth and isolated by adhering the cells to tissue plates at 37°C for 1 h. CD29+ and CD29– peritoneal macrophages were sorted by FACS and transferred to petri dishes. The cells were incubated in 100 µg/ml AF594 hydrazide for 3.5 h at 37°C, followed by 18 h in fluor-free medium, then mixed with AF488-labeled E. coli at a ratio of 50 bacteria per macrophage. After pulsed for 20 min and chased for 30, 60, 90, 120, and 150 min at 37°C, the phagosome maturation after internalization of E. coli was detected by FRET assay, as described above. The bacteria-killing assay with E. coli, GBS, and S. aureus in peritoneal macrophages was also performed, as described above.

Statistical analysis

Statistical significance was analyzed with two-tailed Student’s t test. Values of p < 0.05 were considered significantly different between comparing samples.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Phagocytosis in integrin β1-deficient macrophages

To investigate the function of integrin β1 in macrophage phagocytosis and phagosome maturation, we crossed CD29f/f mice with LysM-Cre transgenic mice to generate β1 integrin-deficient macrophages. The number of macrophages derived from cultured bone marrow cells in CD29f/fLysM-Cre mice was comparable to that in CD29f/f mice (data not shown). We examined integrin β1 deletion in BMM{phi} from CD29f/fLysM-Cre mice. Compared with control cells, BMM{phi} from CD29f/fLysM-Cre mice contained a population (~50%) that did not express CD29, indicating that LysM-Cre induced deletion in these cells (Fig. 1A). To examine the role of CD29 in macrophage function, we purified the CD29– BMM{phi} by FACS sorting (Fig. 1). Deletion of CD29 in BMM{phi} did not affect the expression of integrins β2 (CD18), {alpha}4 (CD49d), and {alpha}M (CD11b) (Fig. 1B). However, the expression of integrin {alpha}5 (CD49e) on integrin β1-deficient macrophages was slightly reduced (Fig. 1B). These results demonstrate that loss of integrin β1 expression on macrophages had limited effect on cell surface expression of other integrins.


Figure 1
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  		FIGURE 1. Expression of integrins on CD29–/– BMM{phi}. A, FACS profiles of sorted integrin β1-deficient BMM{phi}. BMM{phi} prepared from CD29f/f and CD29f/fLysM-cre mice were stained with anti-CD11b and anti-CD29 and subjected to FACS sorting. The sorted CD29– and CD29+ BMM{phi} were used in all of the experiments, except those in Fig. 5. B, Surface expression of other integrins on integrin β1-deficient BMM{phi}. CD29–/– cells were stained with anti-CD11b, CD18, anti-CD49d, and anti-CD49e, and analyzed by FACS. Numbers are the MFI for each file.

 
Integrins are known phagocytic receptors. Integrins {alpha}Mβ2 (CD11b/CD18) and {alpha}xβ2 (CD11c/CD18) function as phagocytic receptors in complement-mediated phagocytosis (1, 3). We first determined whether deletion of integrin β1 affects Fc{gamma}R-mediated and complement-mediated phagocytosis. In phagocytosis of IgG-coupled latex beads, IgG-opsonized SRBCs, or C3-opsonized zymosan, integrin β1-deficient macrophages internalized particles largely comparable to control cells (Fig. 2, A and B), indicating that neither Fc{gamma}R-mediated nor complement-mediated phagocytosis depends on integrin β1. We then examined the phagocytosis of various strains of bacteria opsonized with normal mouse serum by integrin β1-deficient and control BMM{phi}. Macrophages lacking β1 integrin phagocytosed E. coli at a level that was not significantly different from that of control macrophages (Fig. 2C). However, integrin β1-deficient macrophages phagocytosed significantly less GBS and S. aureus (~40% reduction) (Fig. 2C). This impairment was not due to defective surface binding of bacteria to these macrophages because the mean fluorescence intensity (MFI) of bound bacteria on control and CD29-deficient macrophages without quenching was comparable (Fig. 2D). These results indicate that efficient phagocytosis of GBS and S. aureus critically depends on integrin β1, and suggest that FcR and complement receptor-mediated phagocytosis cannot compensate for the loss of integrin β1.


Figure 2
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  		FIGURE 2. Phagocytosis in integrin β1-deficient macrophages. A, Fc{gamma}R-mediated phagocytosis by integrin β1-deficient BMM{phi}. BMM{phi} were cultured on glass coverslips and incubated with IgG-coupled latex beads or IgG-opsonized SRBCs at a ratio of 1:10 for 15–30 min at 37°C to assay phagocytosis. B, Complement-mediated phagocytosis in integrin β1-deficient macrophages. FITC-labeled zymosan particles (5.4 x 107) were opsonized with 150 µl of normal mouse serum at 37°C for 30 min and mixed with macrophages at a ratio of 1:10. Phagocytosed FITC-zymosan particles were counted under a fluorescence microscope. C, Phagocytosis of bacteria by the defective phagosome maturation in β1-deficient BMM{phi} may be due to a role of integrin β1 in the development of macrophages in vitro. BMM{phi} from integrin β1-deficient and WT control mice and FITC-labeled bacteria opsonized with normal mouse serum were mixed in suspension for 30 min at 37°C at a ratio of 1:100 (cell/bacteria). Phagocytosis was assessed by detecting the MFI of the cells after quenching by FACS. Data are mean ± SD of triplicate determinations and are representative of three independent experiments. *, p < 0.01. D, Bacterial binding on β1-deficient BMM{phi}. Experiments were performed as in C without quenching. p > 0.05 in all groups.

 
Impaired phagosome maturation in integrin β1-deficient macrophages

Phagocytosed bacteria are contained within phagosomes that mature into phagolysosomes. Colocalization of phagosomes and lysosomal markers has often been used to indicate phagosome maturation. LysoTracker selectively labels late endosomes and lysosomes, and colocalizes with LAMP (25, 26). We monitored the maturation of phagosomes containing AF488-labeled E. coli by their ability to colocalize with Lysotracker Red over time under confocal fluorescence microscopy. The percentage of colocalization between these two colored markers (weighted for integrated brightness) within each cell was quantitatively analyzed by Metamorph offline software (27, 28). Whereas the percentage of E. coli colocalized with Lysotracker Red reached 42% in WT macrophages at 1 h, colocalization was <20% in integrin β1-deficient macrophages at this time point (Fig. 3, A and B). Similarly, dramatically reduced colocalization of AF488-labeled E. coli with Lysotracker Red in β1 integrin-deficient BMM{phi} was observed at 90, 120, and 150 min when compared with WT macrophages (Fig. 3, A and B). These results suggest that phagosome maturation in integrin β1-deficient macrophages was severely impaired. The lack of colocalization is not a consequence of reduced E. coli uptake because comparable rates of E. coli phagocytosis were observed in integrin β1-deficient and control macrophages (Fig. 2C).


Figure 3
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  		FIGURE 3. Impaired phagosome/lysosome fusion in integrin β1-deficient macrophages. A, Confocal images showing the colocalization of AF488-labeled E. coli with Lysotracker Red in integrin β1-deficient macrophages. BMM{phi} preadhered to coverslips were preloaded with Lysotracker Red and incubated with opsonized AF488-E. coli at a ratio of 1:50 (cell/bacteria) for 20 min at 37°C and chased for 30, 60, 90, 120, and 150 min at 37°C. The colocalization of AF488-E. coli with Lysotracker Red at various time points was examined by laser confocal microscopy. Shown are representative images of two independent experiments. B, Comparison of rate and extent of colocalization of phagosome with lysosome. The percentages of green E. coli-containing phagosomes that were colocalized with Lysotracker Red were measured using Metamorph offline software by randomly scanning >10 cells in each test group in two independent experiments. Data are mean + SD and representative of two experiments. C, Acquisition of endosome and lysosome membrane markers, Rab5, LAMP-1, and LAMP-2 in latex bead-containing phagosomes from integrin β1-deficient and control BMM{phi}. After pulsing with latex beads for 15 min and chasing for 60 or 120 min at 37°C, BMM{phi} were washed and scraped in cold PBS. The phagosomes were then isolated by flotation on a sucrose step gradient. Equivalent amounts of phagosomal proteins were loaded onto 10% SDS-polyacrylamide gels. Rab5, LAMP-1, and LAMP-2 from bead-containing phagosomes were identified by Western blot. Data are representative of three independent experiments. D, Rab5 staining around endocytosed IgG beads. BMM{phi} from integrin β1-deficient and control macrophages containing phagocytosed FITC beads were fixed and stained with anti-Rab5, followed by AF594-labeled anti-mouse Ig.

 
During maturation, phagosomes sequentially acquire membrane markers of early endosomes, late endosomes, and lysosomes. Recent data suggest that LAMP-1 and LAMP-2, predominantly expressed on lysosomes and phagolysosomes, are involved in phagosome maturation (29). To further confirm the defective phagosome maturation in integrin β1-deficient macrophages, we examined the expression of LAMP-1 and LAMP-2 in purified phagosomes from integrin β1-deficient and control macrophages. BMM{phi} were pulsed and chased with latex beads, and phagosomes were isolated using sucrose gradient centrifugation. LAMP-1 and LAMP-2 from bead-containing phagosomes were identified by Western blot. As expected, LAMP-1 and LAMP-2 were strongly expressed in bead-containing phagosomes from control macrophages (Fig. 3C). In contrast, their expression was much reduced in bead-containing phagosomes isolated from integrin β1-deficient macrophages (Fig. 3C). We then examined the expression of Rab5, an early endosome marker (30), in bead-containing phagosomes. The expression of Rab5 was slightly reduced in bead-containing phagosomes from integrin β1-deficient macrophages when compared with control cells (Fig. 3C). We further determined whether this slight reduction of Rab5 was due to less acquisition of Rab5 per phagosome. As shown in Fig. 3D, Rab5 staining around phagocytosed IgG beads in integrin β1-deficient macrophages was similar to that in control macrophages, suggesting that the lower Rab5 protein levels in bead-containing phagosomes from integrin β1-deficient macrophages are most likely due to the slightly reduced phagocytosed IgG beads (see Fig. 2A), but not impaired Rab5 acquisition on individual phagosomes. These results provide additional evidence showing impaired phagosome maturation in macrophages lacking integrin β1.

In addition to the above methods using fluorescent tracers and measuring the expression of phagosome-associated LAMP-1 and LAMP-2 to assess the rate of phagosome maturation, a FRET-based fusion assay has been developed to assess phagosome maturation (15, 31). To further investigate the defective phagosome maturation in integrin β1-deficient macrophages, we performed FRET assays using confocal microscopy to quantitatively assess the rate and extent of fusion between lysosomes and phagosomes. The assay measures the molecule proximity between a donor fluor (AF488-SE or FITC) from the phagocytosed particles and an acceptor fluor (AF594 hydrazide) that have been endocytosed and chased into lysosomes. The FRET signal (the energy transfer efficiency) represents the percentage of increase in donor intensity after acceptor photobleaching. The results showed a rapid increase in FRET signal in a time-dependent manner in WT macrophages, but a dramatically reduced FRET signal in integrin β1-deficient macrophages following uptake of AF488-labeled E. coli (Fig. 4A). The reduced FRET signal in integrin β1-deficient macrophages was due to both reduced percentages of FRET-positive regions and FRET efficiency (Fig. 4B). These data further demonstrate that phagosome maturation is defective in integrin β1-deficient BMM{phi}.


Figure 4
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  		FIGURE 4. Defective phagosome maturation and bactericidal activity in integrin β1-deficient macrophages. FRET assay measuring the access of a phagosome-associated donor fluor (AF488 or FITC) to a lysosome-localized fluor (AF594) was used to quantify phagosome/lysosome fusion following the uptake of opsonized particles. After uptake for 20 min and a chase of 30, 60, 90, 120, or 150 min at 37°C, FRET was measured by laser confocal microscopy using the FRET Acceptor Bleaching program, which verifies the donor molecule’s loss of intensity when in close proximity to an acceptor molecule, before and after selective bleaching of the acceptor. A, Representative images from FRET assay showing the fusion of AF488-labeled E. coli with AF594-labeled lysosome in integrin β1-deficient and control BMM{phi} or peritoneal macrophages. B and C, Comparison of the percentage of FRET-positive regions and FRET efficiency between integrin β-deficient and control BMM{phi} (B) or peritoneal macrophages (C) after phagocytosis of E. coli. The energy transfer efficiency was quantified, as described in Materials and Methods. The percentages of FRET-positive regions were quantified by randomly counting over 10–15 areas within one cell and counting >10 cells in each tested group in three or more independent experiments. D, Defective bactericidal activity in integrin β1-deficient macrophages. BMM{phi} were mixed with opsonized bacteria for 15 min at 37°C at a ratio of cell to bacteria 1:50. After a 90-min chase at 37°C, the intracellular bacteria were released by lysing the macrophages and counted by culturing on agar plates. Data are mean ± SD from triplicate samples and are representative of three independent experiments. E and F, Comparison of FRET efficiency between integrin β1-deficient and control BMM{phi} after phagocytosis of AF488-labeled IgG beads (E) or opsonized FITC-labeled zymosan particles. The experiments were performed as in A–C. *, p < 0.01; **, p < 0.05.

 
The defective phagosome maturation in β1-deficient BMM{phi} may be due to a role of integrin β1 in the development of macrophages in vitro. To exclude this possibility, we further examined phagosome maturation in peritoneal macrophages from CD29f/f or CD29f/fLysM-Cre mice. Similar to BMM{phi} (Fig. 1), ~50% of elicited peritoneal macrophages from CD29f/fLysM-Cre mice were CD29– (data not shown). CD29+ and CD29– peritoneal macrophages were sorted by FACS and tested for phagosome maturation using FRET assays. The FRET signal generated between phagocytosed AF488-SE-labeled E. coli and acceptor fluor (AF594 hydrazide) was dramatically reduced in integrin β1-deficient peritoneal macrophages (Fig. 4A). Furthermore, the percentages of FRET-positive regions and FRET efficiency were both reduced in the absence of integrin β1 in these macrophages (Fig. 4C), suggesting that the defective phagosome maturation in β1-deficient BMM{phi} is not simply due to a role of integrin β1 in the development of macrophages in vitro.

Defective intracellular killing of bacteria in integrin β1-deficient macrophages

Lysosomes containing diverse acid hydrolases are responsible for the degradation of ingested material in macrophages. The defective phagosome maturation in integrin β1-deficient macrophages suggested that these cells might have impaired intracellular killing of ingested bacteria. We determined intracellular bacteria clearance in integrin β1-deficient macrophages. Macrophages lacking integrin β1 contained significantly higher numbers of live E. coli, S. aureus, and GBS than control cells (p < 0.01) (Fig. 4D). Given the fact that integrin β1-deficient macrophages phagocytosed less S. aureus and GBS than control cells (Fig. 2B), the increased numbers of live bacteria in integrin β1-deficient macrophages further indicate that these cells could not efficiently kill the bacteria intracellularly. These results further support that phagosome maturation is defective in integrin β1-deficient peritoneal macrophages.

Impaired maturation of phagosomes from FcR- but not complement receptor-mediated phagocytosis in integrin β1-deficient macrophages

The above FRET assays using opsonized E. coli could not define the phagosome types that depend on integrin β1 for their maturation. To test whether phagosomes derived from FcR- or complement receptor-mediated phagocytosis depend on integrin β1 for their maturation, we performed FRET assays using AF488-labeled IgG beads or opsonized FITC-labeled zymosan particles. Maturation of phagosomes from FcR-mediated phagocytosis in integrin β1-deficient macrophages was impaired (Fig. 4E). In contrast, maturation of phagosomes from complement receptor-mediated phagocytosis in integrin β1-deficient macrophages was comparable to that in control cells (Fig. 4F). These results demonstrate that phagosomes derived from FcR- or complement receptor-mediated phagocytosis differ in their dependence on integrin β1 for their maturation.

Integrin β1 regulates phagosome maturation through Rac expression

A recent study demonstrates that Rho GTPase regulates the maturation of phagosomes containing apoptotic cells, but not opsonized cells in macrophages (32), suggesting that other Rho GTPase family members, including Rac1, Rac2, and Cdc42, may be involved in the maturation of phagosomes containing opsonized cells. Our previous study has found that integrin β1-deficient dendritic cells (DCs) fail to efficiently prime T lymphocytes and express reduced levels of Rac1, Rac2, and Cdc42 (19), suggesting that integrin β1 not only activates Rac1, Rac2, and Cdc42, but also positively regulates their expression in DCs. These results raised the possibility that integrin β1 also regulates Rac1, Rac2, and Cdc42 expression in macrophages. To test this, we examined Rac1, Rac2, and Cdc42 expression in integrin β1-deficient BMM{phi} with or without bacterial stimulation. Total cell lysates from purified β1-deficient and control BMM{phi} stimulated with E. coli were blotted for total Rac1, Rac2, and Cdc42 expression. As expected, control macrophages expressed abundant levels of Rac1, Rac2, and Cdc42 before and after simulation with E. coli (Fig. 5A). In contrast, the expression of total Rac1, Rac2, and Cdc42 proteins in integrin β1-deficient macrophages was severely reduced with or without E. coli stimulation (Fig. 5A). These results suggest that the impaired maturation of phagosomes containing opsonized bacteria in integrin β1-deficient macrophages may be caused by the reduced expression of Rac1, Rac2, and/or Cdc42.


Figure 5
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  		FIGURE 5. Reduced Rac1, Rac2, and Cdc42 expression in integrin β1-deficient macrophages. A, Expression of total Rac1, Rac2, and Cdc42 proteins in integrin β1-deficient or control BMM{phi} before and after stimulation with E. coli for indicated time, as determined by Western blot analysis. The cells lysates were fractionated by 12% SDS-PAGE gel. Expression of actin serves as loading control. B, Ectopic expression of Rac1 and Cdc42 in integrin β1-deficient BMM{phi} as detected by Western blot. The pMI vector expressing hCD2 and mouse WT Rac1 and Cdc42 cDNA bicistronically were used to generate retroviruses. BMM{phi} from CD29f/f or CD29f/fLysM-Cre mice were infected with retroviruses at days 2 and 3 of the culture. Infected hCD2+ and CD29+ or CD29– BMM{phi} were sorted by FACS for Western blot analysis. Expression of actin serves as loading control. C, Restoration of the defective phagocytosis by ectopic expression of Rac1 and Cdc42 in integrin β1-deficient macrophages. Infected hCD2+ and CD29+ or CD29– BMM{phi} after sorting by FACS were mixed with FITC-labeled S. aureus or GBS at a ratio of 1:100 at 37°C for 30 min. Phagocytosis was assessed by detecting the MFI of the cells by FACS. Data are representative of three experiments. *, p < 0.01.

 
We then tested whether ectopically expressed Rac1 or Cdc42 could restore the defective phagocytosis of bacteria and phagosome/lysosome fusion in integrin β1-deficient macrophages. Because Rac1 and Rac2 share 92% homology and are functional redundant (22), we reasoned that expression of Rac1 would most likely rescue the reduced expression of both Rac1 and Rac2 in integrin β1-deficient macrophages. Integrin β1-deficient and control BMM{phi} expressing retroviral vector pMI (24)-delivered Rac1 or Cdc42 were sorted by FACS based on hCD2 expression. The expression levels of Rac1 and Cdc42 in integrin β1-deficient macrophages were restored to at least those in control macrophages (Fig. 5B). We first examined whether Rac1 or Cdc42 expression could restore the reduced phagocytosis of GBS and S. aureus in integrin β1-deficient macrophages. The results showed that expression of either Rac1 or Cdc42 restored defective phagocytosis of S. aureus, but not GBS (Fig. 5C), suggesting that phagocytosis of these two bacteria differentially depends on Rac1 and Cdc42 most likely through distinct phagocytic receptors.

Phagosome maturation depends on efficient fusion of phagosomes with early endosomes, late endosomes, and lysosomes. This process is facilitated by movement of phagosomes along microtubules and requires actin assembly at the phagosomal membrane (33). We tested whether there is a defect in F-actin assembly around the phagosomes in integrin β1-deficient macrophages and whether ectopically expressed Rac1 or Cdc42 could restore defective F-actin assembly. The colocalization of phagosomes containing AF488-labeled E. coli with F-actin stained by rhodamine-phalloidin was quantified. We observed a 2.7-fold decrease in phagosome-associated F-actin in integrin β1-deficient BMM{phi} when compared with control cells (Fig. 6, A and B), suggesting that actin accumulation around phagosomes in integrin β1-deficient macrophages is impaired. Interestingly, ectopically expressed Rac1, but not Cdc42, restored the defective F-actin assembly in the periphagosomal region after internalization of E. coli in integrin β1-deficient macrophages (Fig. 6, A and B).


Figure 6
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  		FIGURE 6. Integrin β1 regulates phagosome maturation through Rac1 expression. A, Defective F-actin polymerization in the periphagosomal region and restoration by ectopic Rac1 expression in integrin β1-deficient BMM{phi} during phagosome maturation. Polymerization of cellular actin was measured by phalloidin binding. Macrophages after internalization of AF488-labeled E. coli for 30 min were fixed and permeabilized for 5 min with 0.1% Triton X-100 in PBS and then stained with 0.2 µM rhodamine-phalloidine for 30 min on ice. Images showing the staining of F-actin and phagosomes were obtained by laser confocal microscopy. B, The percentages of phagosomes that were colocalized with F-actin were measured by Metamorph offline software. Data are mean ± SD and representative of three independent experiments. C, Effect of ectopic Rac1 and Cdc42 expression on phagosome/lysosome fusion in integrin β1-deficient macrophages. Retrovirus-infected hCD2+ and CD29+ or CD29– BMM{phi} were sorted by FACS and used for FRET assays to determine the rate and extent of phagosome/lysosome fusion. Data are representative of three experiments. *, p < 0.01; **, p < 0.05.

 
We then tested whether ectopically expressed Rac1 or Cdc42 could restore the defective phagosome/lysosome fusion in integrin β1-deficient macrophages. Phagosome/Lysosome fusion in integrin β1-deficient and control BMM{phi} expressing Rac1 or Cdc42 was detected by FRET assay. As shown in Fig. 6C, Rac1 expression readily restored the defective phagosome/lysosome fusion. In contrast, ectopic expression of Cdc42 in integrin β1-deficient macrophages failed to restore defective phagosome maturation (Fig. 6C). These results suggest that integrin β1 regulates phagosome maturation through Rac1/2 expression in macrophages.

Defective phagosome maturation in macrophages lacking Rac1/2

To further determine the role of Rac1 and Rac2 in phagosome maturation, we performed FRET assays using macrophages from mice conditionally lacking both Rac1 and Rac2 (22). As expected, the phagocytosis of opsonized E. coli and IgG beads by Rac1/2-deficient macrophages was impaired (data not shown). It should be noted in this study because our FRET assay only measures the maturation of those phagosomes that contain labeled particles, the impaired phagocytosis of Rac1/2-deficient macrophages should not skew the results from FRET assay of phagosome maturation. Similar to the impaired phagosome maturation in integrin β1-deficient macrophages, maturation of phagosomes from both phagocytosed E. coli and IgG beads in Rac1/2-deficient macrophages was significantly reduced when compared with that in control macrophages (Fig. 7). These results demonstrate that Rac1/2 are critically involved in phagosome maturation.


Figure 7
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  		FIGURE 7. Defective phagosome maturation in Rac1/2-deficient macrophages. FRET assay using Rac1/2-deficient macrophages measuring the access of a phagosome-associated donor fluor (AF488) to a lysosome-localized fluor (AF594) was performed, as described in Fig. 4. A, Representative images from FRET assay showing the fusion of AF488-labeled E. coli or IgG beads with AF594-labeled lysosome in Rac1/2-deficient and control BMM{phi}. B, Comparison of FRET efficiency between Rac1/2-deficient and control BMM{phi}. p < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we show that macrophages lacking integrin β1 exhibit defects in the phagocytosis of GBS and S. aureus and in phagosome maturation after internalization of opsonized E. coli and IgG beads. These defects may be attributed to the function of integrin β1 in regulating Rac1/2 and Cdc42 expression because integrin β1-deficient macrophages express reduced levels of Rac1/2 and Cdc42. Furthermore, ectopic expression of Rac1 or Cdc42 in these cells restored phagocytosis of S. aureus, and ectopic expression of Rac1 restored their defective phagosome maturation. These defects were not caused by impaired development of bone marrow-derived integrin β1-deficient macrophages in vitro, because similar defects were observed in peritoneal macrophages lacking integrin β1 expression. The defective phagocytosis and phagosome maturation of integrin β1-deficient macrophages were not due to a general defect in macrophage development. Integrin β1-deficient macrophages have normal FcR-mediated and complement-mediated phagocytosis. Furthermore, phagocytosis of E. coli by integrin β1-deficient macrophages is normal.

Integrins as heterodimeric cell surface receptors play important function in cell trafficking and cellular adhesion. Integrin signaling is critical for cellular functions such as firm adhesion, cell spreading, chemotaxis, the production of reactive oxygen intermediates, and the release of antimicrobial granule proteins (17, 34). In addition, integrins may serve as phagocytic receptors for microbial pathogens. Integrin {alpha}Mβ2 (complement receptor 3) is one of the best-characterized phagocytic receptors, binding to particles opsonized with complement C3bi (1, 3, 10). Our previous data have shown that mindin, a highly conserved extracellular matrix protein, is a novel pattern recognition molecule for microbial pathogen and functions as an opsonin in the phagocytosis of bacteria (35). Mindin-deficient macrophages also exhibit defective phagocytosis of GBS and S. aureus (35). Given that mindin serves as a ligand for {alpha}Mβ2, {alpha}4β1, and {alpha}5β1 integrins on neutrophils and DCs (18, 19), the defective phagocytosis of GBS and S. aureus displayed by both mindin-deficient and integrin β1-deficient macrophages suggests that the interaction between mindin and integrin β1 is critical for efficient phagocytosis of these bacteria. The dependence of macrophage phagocytosis on mindin and integrin β1 cannot be compensated by other extracellular matrix proteins, integrins, and FcR.

Our data have defined β1 integrin as a key signaling receptor in regulating phagosome maturation and microorganism clearance in macrophages. Phagosome maturation is defined as the process by which phagosomes change their functional capacity as they age within cells. The main inducer of these alterations in phagosome behavior is thought to be the sequential process of fusion, which takes place first with early endosome organelles and then with the late endosomes and lysosomes (6, 36, 37). The detailed molecular mechanisms responsible for the regulation of phagosome maturation pathways are largely unknown. Recent studies suggest that phagosome maturation may be regulated by signals from TLRs through the adaptor protein MyD88 and the MAPK p38. When TLR signaling is engaged by ligands present in the cargo, an inducible mode of phagosome maturation occurs (6, 14). However, another recent study found no differences in the rate of phagolysosomal fusion of phagosomes containing mannose- or IgG-coated beads in the presence or absence of ligands that stimulate TLRs (15). Thus, the role of TLRs in regulating phagosome maturation needs further investigation.

Rho-related small GTPases, including Rho, Rac, and Cdc42, are known to play a central role in adhesion, cell shape formation, and motility in a variety of mammalian cell types (38). They regulate actin cytoskeletal reorganization through various effector proteins, which interact with active GTP-bound forms of the rho family (38, 39). It has been well established that Rac1- and Cdc42-induced actin assembly plays essential roles during the formation of phagocytic cups in FcR-mediated phagocytosis (7, 8, 9, 10). However, the roles of these Rho GTPases in regulating phagosome maturation after internalization of bacteria were not clear. A very recent study demonstrates that Rho GTPase regulates the maturation of phagosomes containing apoptotic cells, but not opsonized cells in macrophages (32), suggesting that other Rho GTPase family members Rac1, Rac2, or Cdc42 may be involved in the maturation of phagosomes containing opsonized bacteria. Our data have shown that Rac1/2 is critically required for the maturation of phagosomes containing serum-opsonized bacteria or IgG-conjugated beads.

Our results suggest that integrin β1 regulates phagosome maturation through Rac expression. This regulation can be directly or indirectly. Furthermore, our results do not exclude the possibility that integrin β1 also regulates other aspects of phagocytosis in macrophages. Phagosomal maturation requires drastic remodelling of the phagosomal membrane and contents. There is compelling evidence that phagosome maturation not only depends on microtubules, but also involves the actin cytoskeleton (5, 33). Mature phagosomes were reported to be often surrounded by an actin-rich cytoplasm (40). Taunton et al. (41) demonstrated that endosomes and lysosomes are propelled by the formation of actin comet tails. Furthermore, actin was detectable as a component of fully formed phagosomes (4, 42), and numerous actin-binding proteins, including annexins, {alpha}-actinin, and coronin, are also found to associate with phagosomes (42, 43). We found that actin accumulation (or rearrangement) around phagosomes was impaired in integrin β1-deficient macrophages. This suggests that reduction of Rac1/2 in integrin β1-deficient macrophages may impair the process of phagosome/lysosome fusion through a decreased phagosome-associated actin polymerization. Our data show that ectopically expressed Rac1, but not Cdc42, not only restored the defective F-actin assembly in the periphagosomal region after internalization of E. coli, but also restored phagosome maturation as assessed by FRET assay in integrin β1-deficient macrophages. Furthermore, Rac1/2-deficient macrophages also exhibit a similar defect in maturation of phagosomes from phagocytosis of E. coli and IgG beads. These results suggest that integrin β1-mediated signaling plays a critical role in regulating phagosome maturation through Rac expression in macrophages.

Given the reduced expression of Rac1/2 and Cdc42 and impaired F-actin accumulation around the phagosome region in macrophages lacking integrin β1, one might expect that integrin β1-deficient macrophages should have defective FcR-mediated phagocytosis. But surprisingly, we have observed a normal phagocytosis of IgG-coupled beads or IgG-opsonized SRBCs by these macrophages, which is in contrast to macrophages lacking both Rac1/2. This may indicate that residual levels of Rac1 and Cdc42 in integrin β1-deficient macrophages are sufficient to induce phagocytic cup formation during FcR-mediated phagocytosis. In summary, the observations made in this study demonstrate that integrin β1 plays an essential role in regulating phagosome maturation. Deeper insight into the signaling process that regulates Rac expression through integrin β1 and that connects the signals from Rac to membrane traffic of phagosomes will be important to our understanding of the molecular machinery in phagosome maturation.


   Acknowledgments
 
We thank Mike Cook and Lynn Martinek in the Flow Cytometry Facility of Duke University Medical Center for FACS sorting, and Nu Zhang, Heather H. Pua, Claire Gordy, and Ivan Dzhagalov for critical reading of this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by National Institutes of Health Grants AI054685 and AI061364 to Y.-W.H. Back

2 Address correspondence and reprint requests to Dr. You-Wen He, Box 3010, Department of Immunology, Duke University Medical Center, Durham, NC 27710. E-mail address: he000004{at}mc.duke.edu Back

3 Abbreviations used in this paper: GBS, group B streptococcus; AF, Alexa Fluor; BMM{phi}, bone marrow-derived macrophage; DC, dendritic cell; FRET, fluorescence resonance energy transfer; hCD2, human CD2; LAMP, lysosomal-associated membrane protein; MFI, mean fluorescence intensity; WT, wild type. Back

Received for publication April 30, 2007. Accepted for publication December 6, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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